Increasing electro-gene transfer of nucleic acid molecules into host tissue

ABSTRACT

A method of delivering a pharmaceutical agent, such as a nucleic acid molecule, to a vertebrate host is disclosed which comprises combining the synergistic steps of electrical stimulation with administration of a biologically active amount of hyaluronidase. Additionally, formulations which comprise hyaluronidase and a pharmaceutical agent, such as a nucleic acid molecule, are disclosed. The hyaluronidase preparation is preferably administered prior to or simultaneous with the pharmaceutical agent and in conjunction with an applied electrical stimulation, thus affecting increased transfer of the population of a pharmaceutical agent into the target tissue when compared to the affect of electrical stimulation alone. The combination of hyaluronidase administration and electrostimulation results in a substantial increase in the transfer of the pharmaceutical agent, such as a nucleic acid molecule, to the target vertebrate host tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit, under 35 U.S.C. §119(e), to U.S.provisional application 60/333,338 filed Nov. 26, 2001.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable

REFERENCE TO MICROFICHE APPENDIX

Not Applicable

FIELD OF THE INVENTION

The present invention relates to methods of increasing the efficiency ofelectrical-based transfer of pharmaceutical agents, such as nucleic acidmolecules, into a vertebrate host, such as a human or animal host. Themethods of the present invention further concern administration of abiologically effective amount of hyaluronidase prior to or simultaneouswith administration of the respective pharmaceutical agent andapplication of an electrical stimulation, thus affecting increasedtransfer of a population of the pharmaceutical agent into the targettissue when compared to the affect of electrical stimulation alone. Suchmethodology represents an improved efficiency of transfer and expressionof nucleic acid molecules with the target tissue of the respective host.The present invention may also be used in conjunction with othercompounds such as proteins and peptides.

Formulations comprising hyaluronidase and a pharmaceutical agent arealso disclosed. Such formulations allow for a single administration ofthese two components in conjunction with an appropriate electricalstimulus.

BACKGROUND OF THE INVENTION

Studies have shown that applied electrical energy can affect abiological membrane, in that a sufficient application of energyincreases the permeability of the membrane and thus allows solutions todiffuse through a membrane or tissue more readily to achieve a desiredeffect. Generally, electrical or electromagnetic stimulation effectshave been explained with reference to one or more of iontophoresis,electrophoresis or electroporation (collectively “electricalstimulation” or “electrostimulation”, or in the context of the transferof nucleic acid molecules, “electro-gene transfer”, or “EGT”), which areeither different forms of electrical stimulation or different ways tointerpret the effects of an electromagnetic field. Iontophoresisgenerally concerns the introduction of an ionized substances through anintact membrane such as the skin, by application of a direct electriccurrent. The current presumably entrains the ions and/or increases ionmobility in the tissue. Electrophoresis concerns the migration of ionsin a fluid or gel under influence of an electric field. Inelectroporation, an electric field (often pulsed) and the associatedinduced current, induce microscopic pores to form in a membrane,typically a cell membrane. These pores are commonly called“electropores” and the process of forming them is electroporation. Apotential application of electroporation is that solutions such aspharmaceutical agents, molecules, ions, and/or water can pass morereadily from one side of the membrane to the other through theelectrically generated pores. The pores preferably persist temporarilyduring application of the field. After application of the field, thepores should close or heal within a short period of time. However, thehealing time is dependant on the amplitude and duration of theelectrical stimulation, and it is possible to damage tissue permanentlyby application of too high an instantaneous power level and/or too longa duration of stimulation. The damage could be due to formation ofuntenably large or numerous pores, or resistive heating of the tissue,or both.

Electrically induced pores have been observed and studied to a degree,in vitro, where cells in a solution are substantially independent of oneanother and are exposed to view. The situation is not readily observedin vivo. If an observation could be made at a particular site and on themicroscopic scale that might be most pertinent, it would likely beatypical due to the effects of field and current density variations inthe tissue or as induced by the apparatus employed to make the 0.30observation.

Genetic and immunological therapies are candidates for theelectroporation of tissues. In electrical stimulation of tissues,contact and non-contact apparatus are possible. In a contact apparatus,a signal is applied by physically contacting a target tissue site usingconductive electrodes attached on opposite sides of the target site. Ina non-contact apparatus, an electric or magnetic field can be generatedusing electrodes or coils that are likewise disposed on opposite sidesof the site. In the contact example, the tissue may have a reactivecomponent (capacitance or inductance) and the conductivity of the tissuemay change over time due to the effects of the application of energy(e.g., due to heating), but in general the electrical response of thetissue is according to Ohm's law.

Direct plasmid DNA gene transfer is currently the basis of many emergingtherapeutic strategies as it avoids the potential problems associatedwith viral genes and lipid particles (e.g., see van Deutekom et al.,1998; Mol. Med. Today 4: 214-220; Treco and Selden, 1995, Mol. Med.Today 1: 314-321). Skeletal muscle-borne plasmids have been expressedefficiently over months or years in immunocompetent hosts (e.g., seeWolff et al., 1992, Hum. Mol. Genet. 1: 363-369; Davis et al., 1993;Manthorpe et al., 1993, Hum. Gene Ther. 4: 419-431) leading to transgeneexpression and physiological or therapeutic responses, such as vaccinaland anti-inflammatory response or hematocrit (Hct) increase (e.g., seeDavis et al., 1996; Proc. Natl Acad. Sci. USA 93: 7213-7218; Kessler etal, 1996; Proc Natl Acad Sci USA. 93: 14082-14087; Kreiss et al, 1999,J. of Gene Med. 1: 245-250; Levy et al., 1996, Gene Therapy 3: 201-211;Miller et al., 1995, Gene Therapy 2: 736-742; Song et al., 1998; J.Clin. Invest. 101: 2615-2621; Trypathy et al., 1996, PNAS 93:10876-10880. However, the high individual variability of foreign geneexpression, and the low level of therapeutic protein expression,particularly in large animals (see Jiao et al., 1992, Hum. Gene Therapy3: 21-33) are limiting factors to the use of naked DNA injection forclinical application. Nonetheless, the development of an efficienttransfer method for plasmid DNA would be ideal for applications in avariety of diseases.

U.S. Pat. No. 6,110,161, issued Aug. 29, 2000 to Mathiesen et al. (seealso, WO 98/43702 and Mathiesen, 1999, Gene Therapy 6: 508-514) disclosein vivo electrostimulation of skeletal muscle within a calculatedelectric field strength ranging from about 25 V/cm to about 250 V/cm.

WO 99/01158, WO 99/01157 and WO 99/01175 disclose the use of low voltagefor a long duration to promote in vivo electrostimulation of naked DNA.An electric field strength or voltage gradient of about 1 V/cm to about600 V/cm is disclosed, depending upon the target tissue. Thisencompasses a relatively expansive range from minimal effect topotentially injurious levels. However, even higher voltage gradientshave been proposed.

U.S. Pat. No. 5,810,762, U.S. Pat. No. 5,704,908, U.S. Pat. No.5,702,359, U.S. Pat. No. 5,676,646, U.S. Pat. No. 5,545,130, U.S. Pat.No. 5,507,724, U.S. Pat. No. 5,501,662, U.S. Pat. No. 5,439,440 and U.S.Pat. No. 5,273,525 disclose electroporation/electrostimulationmethodology and related apparatus wherein it is suggested that a usefulelectrical field strength range within the respective tissue is fromabout 200 V/cm to about 20 KV/cm. U.S. Pat. Nos. 5,968,006 and 5,869,326further suggest that electric field strengths as low as 100 V/cm areuseful for certain in vivo electrostimulation procedures.

-   Jaroszeski et al. (1999, Advanced Drug Delivery Reviews 35: 131-137)    review the present landscape of in vivo electrically mediated gene    delivery techniques. The authors emphasize previous success with    delivery of chemotherapeutic agents to tumor cells and discuss some    of the early results in this field.-   Titomirov et al.(1991, Biochem Biophys Acta 1088: 131-134) delivered    two plasmid DNA constructs subcutaneously followed by electrical    stimulation of skin folds, generating an electric field strength    from 400 V/cm to 600 V/cm.-   Heller et al. (1996, FEBS Letters 389: 225-228) delivered plasmid    DNA expressing two reporter genes to rat liver tissue by generation    of high voltage pulses (11.5 KV/cm) rotated through a circular array    of electrodes.-   Nishi et al. (1996, Cancer Res. 56: 1050-1055) delivered plasmid DNA    expressing a reporter gene to rat brain tissue. The authors utilized    an electric field strength of approximately 600 V/cm.-   Zhang et al. (1996, Biochem. Biophys. Res. Comm. 220: 633-636)    delivered plasmid DNA transdermally to mouse skin with 120V pulses    to the skin folds wherein the distance between the electrodes was    only about 1 mm.-   Muramatsu et al. (1997, Biochem. Biophys. Res. Comm. 223:45-49)    reported transfection of mouse testis cells with plasmid DNA via 100    V pulses with a 10 mS pulse duration.-   Rols et al. (1998, Nature Biotechnology 16(2): 168-171) reported    transfection of mouse tumor cells with plasmid DNA by applying    voltages from about 300 to 400 V across a 4.2 mm spacing of the    electrodes.-   Aihara and Miyazaki (1998, Nature Biotechnology 16: 867-870)    reported in vivo expression of β-gal in mouse muscle tissue by    delivering a square waveform pulse (50 mS duration) at constant    voltage (60V) with the distance between the electrodes being 3-5 mm.-   Vicat et al. (2000, Human Gene Therapy 11: 909-916) show that high    voltage (900 V), short pulse (100_S) electrostimulation protocols    result in prolonged expression within targeted cells, in this case    mouse muscle cells.-   Widera et al (2000, J. Immunology 164: 4635-4640) apply 100 volts    over a 5 mm distance with conducting electrodes to deliver hepatitis    B surface antigen, HIV gag and env encoding DNA vaccines in vivo to    mouse and guinea pigs.-   Suzuzki et al. (1998, FEBS Lett. 425: 436-440) apply a voltages of    25, 50 and 100 V to the liver lobe of a rat. The authors found that    8 pulses (50 ms each) of 50 V was optimal for GFP expression.-   Goto et al. (2000, Proc. Natl. Acad. Sci. USA. 97: 354-359) show    delivery of the “A” fragment of diphtheria toxin and the HSV TK gene    to mouse tumors via voltage pulses (with an electric field strength    of approximately 66 V/cm) through a circular array of six needle    electrodes reduces tumor growth in mice.-   Oshima et al. (1998, Gene Therapy 5: 1347-1354) show EGT to rat    corneal endothelium.-   Favre et al. (2000, Gene Therapy 7: 1417-1420) shows that HYAse    enhances adeno-associated virus mediated gene transfer in rat    skeletal muscle by increasing viral diffusion in the injected tissue-   Fromes et al. (2000, Gene Therapy 6: 683-688) show that a mix of    HYAse and collagenase increase adenovirus diffusion in rat    myocardium.-   Batra et al. (1997, J. Biol. Chem. 272: 11736-11743) show inhibition    of retroviral gene transfer to cancer cells by extracellular    components of malignant pleural effusion, and neutralization of this    inhibition by treatment of effusions with HYAse and chondroitinases.-   Dubensky et al. (1984, Proc. Natl. Acad. Sci. USA, 81: 7529-7533)    show that injecting polyoma-plasmid recombinant DNA along with HYAse    and collagenase leads to more uniform transfection of mouse livers    and spleens.

It would be advantageous to identify improved methods ofelectrical-based transfer of pharmaceutical agents into host tissuewhich provide for enhanced and long lasting gene expression without anysignificant tissue alteration. The present invention addresses and meetsthese needs by disclosing methodology which comprises administration ofa biologically effective amount of hyaluronidase in combination withelectrical stimulation to increase the gene transfer and expressionwithin host tissue.

SUMMARY OF THE INVENTION

The present invention relates to a method of delivering a pharmaceuticalagent into a tissue of a vertebrate host which comprises the steps ofadministering a biologically effective amount of hyaluronidase to thetissue of the vertebrate host; administering the pharmaceutical agentproximal to the delivery points of HYAse administration and, applying anelectrical stimulus proximal to the site of administration of thehyaluronidase and the pharmaceutical agent. To this end, the methodologyof the present invention relates to delivery of a pharmaceutical agent,such as a population of nucleic acid molecules (exemplified herein asDNA plasmid molecules), into the tissue of a vertebrate host, whichcomprises a) administering a biologically effective amount ofhyaluronidase to the tissue of the vertebrate host; b) administering thepharmaceutical agent proximal to the delivery site of hyaluronidaseadministration in step a); and, c) applying an electrical stimulusproximal to the delivery points of step a) and step b). This methodologyresults in a substantial increase in delivery, and hence in vivoefficacy, of electrical-based delivery technology.

One aspect of the present invention relates to methods of enhancing theelectro-gene transfer (EGT) of nucleic acid molecules into a hostvertebrate tissue which comprises administering hyaluronidase (HYAse) incombination with a physiologically acceptable EGT protocol, as describedin the above paragraph. The combination of a particular EGT protocolwith a HYAse injection results in increased transfer of nucleic acidmolecules as compared to application of the respective EGT protocolalone.

The present invention also relates to pharmaceutical formulation whichcomprises an effective amount of hyaluronidase and the respectivepharmaceutical agent. A preferred pharmaceutical agent is an effectiveconcentration of nucleic acid molecules, and most preferably abiologically effective concentration of DNA plasmid molecules.

To this end, the present invention relates to methods of enhancingelectro-gene transfer (EGT) of nucleic acids into vertebrate tissuewhich comprises administering a biologically effective amount ofhyaluronidase (HYAse) in combination with an EGT treatment.

To this end, the present invention relates to methods of enhancing EGTof nucleic acids into mammalian tissue which comprises administering abiologically effective amount of hyaluronidase (HYAse) in combinationwith an EGT treatment.

The present invention further relates to methods of enhancing EGT ofnucleic acids into mammalian muscle tissue which comprises administeringa biologically effective amount of hyaluronidase (HYAse) in combinationwith a respective pharmaceutical agent in conjunction with an EGTtreatment.

Therefore, a preferred vertebrate target host is a mammal, and anespecially preferred target host includes but is not limited to humansand non-human primates, and may also include any non-human mammal ofcommercial or domestic veterinary importance.

Additionally, while one or more tissue types from the vertebrate hostmay be targeted for the synergistic EGT/HYAse methodology of the presentinvention, a preferred tissue type is muscle tissue, which has beenshown to be a viable target tissue for various electrostimulationprotocols involving gene therapy and/or gene vaccination applications. Apreferred mode of administration for either/or of the gene construct andHYAse is by direct needle injection.

A specific embodiment of the present invention relates to the timing ofHYAse administration in relation to application of the respective EGTtreatment. As shown in FIG. 2, administration of HYAse anywhere from 10minutes to 4 hours results in an increased efficiency in gene transfer.Therefore, it is preferred that administration of HYAse be prior to orconcurrent with application of the EGT treatment. It will be within thepurview of the skilled artisan to optimize a specific EGT treatment witha specific time of HYAse administration with a specific target host,knowing that preinjection of HYAse should increase the efficiency of therespective gene transfer protocol. The ability to administer HYAse justprior to or even in conjunction to EGT lends itself to formulationswhich comprise both HYAse and the respective pharmaceutical agent, suchas a nucleic acid molecule. To this end, the present invention relatesto a formulation which comprises both HYAse and the respectivepharmaceutical agent, such as a nucleic acid molecule, or morepreferably, a biologically effective amount of a DNA plasmid constructwhich expresses the transgene/antigen of interest upon in vivoadministration.

A specific embodiment of the present invention thus relates to theincreasing the efficacy of electro-gene transfer (EGT) of plasmid DNAinto skeletal muscle by preinjecting hyaluronidase (HYAse), whichsignificantly increases the gene transfer efficiency of muscle EGT. Twoconstructs encoding mouse erythropoietin (PCMV/mEPO) and secretedalkaline phosphatase (pCMV/SeAP) were electro injected intramuscularlyin Balb/C mice and rabbits with and without HYAse pretreatment.Preinjection 1 or 4 hr prior to EGT increased EPO gene expression byabout 5 fold in mice and maintained higher gene expression than plasmidEGT alone. A similar increment in gene expression was observed uponpretreatment with HYAse and pCMV/mEPO electroinjection in rabbittibialis muscle. The increment of gene expression in rabbits reached 17fold upon injection of plasmid pCMV/SeAP.

It is an object of the present invention to provide for an enhancementof EGT-based protocols for, in vivo gene transfer into vertebratetissues wherein hyaluronidase (HYAse) is administered in combinationwith a respective pharmaceutical agent and an effective EGT protocol.The combination of a particular EGT protocol with a HYAse injection(preferably prior to, and possibly at or near the time ofelectrostimulation of tissue surrounding the area of nucleic aciddelivery), thus resulting in increased efficiency of gene transfer,expression and/or immunogenicity of a respective gene construct ascompared to application of the respective EGT protocol alone.

It is also an object of the present invention to provide forformulations which comprise both HYAse and a respective pharmaceuticalagent, such as a population of nucleic acid molecules which, upon invivo administration, result in expression of a respectivetransgene(s)/antigen(s).

As used herein, “p.i.” is an abbreviation for—post injection—.

As used herein, “EGT.” is an abbreviation for—electro-gene transfer—,which is used interchangeably with the terms “electrostimulation” and“electrical stimulation.”

As used herein, “HYAse” is an abbreviation for—hyaluronidase—.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B shows dependence of plasmid pCMV/mEPO expression on theconcentration of HYAse preinjected in Balb/c mice. A. Serum EPO levels.B. Hematocrit levels. Groups of 4 mice were injected in quadricepsmuscle with different doses of HYAse 4 hr prior to plasmid EGT. Bloodsamples were collected 10 days p.i. and compared to EGT alone and salinecontrols. Data are the mean ±SD of hematocrit and serum EPO.*Significantly different from EGT alone.

FIG. 2 shows the effect of HYAse preinjection time on plasmid EGTenhancement. Groups of 4 Balb/c mice were preinjected with 36 U of HYAseat different times prior to plasmid EGT. Blood samples were collected 7days p.i. and compared to EGT alone and saline controls. Data are themean ±SD serum EPO.* Significantly different from EGT alone.

FIG. 3A-C shows the long term effect of HYAse injection on EPOexpression. Groups of 4 Balb/c mice were injected with 36 U of HYAse 1hr prior to plasmid EGT. The serum EPO levels of animals injected withdifferent DNA doses were compared at (A) 7, (B) 56, and (C) 120 daysp.i. Data are the mean ±SD serum EPO. *Significantly different from EGTalone.

FIG. 4A-C shows histological analysis of HYAse injected mousequadriceps. Mice quadriceps were injected with 36 U of HYAse and tissueswere analyzed (A) 3, (B) 7, and (C) 30 days p.i.

FIG. 5A-B shows the effect of HYAse injection on plasmid EGT in rabbits.180 U of HYAse were injected in the tibilias muscle 40 min. prior toplasmid EGT. Rabbits were injected with either 200 μg of plasmidpCMV/mEPOopt or with 200 μg of plasmid pCMV/SeAP. Blood samples werecollected 4 days p.i. and serum EPO (A) and SeAP (B) levels weremeasured and compared to those detected in animals treated with plasmidEGT alone. Data are the mean ±SD as measured in four rabbits.*Significantly different from EGT alone.

FIG. 6 shows expression of β-galactosidase after plasmid pCMV/β-gal/NLSEGT with or without HYAse preinjection. 360 U of HYAse were injected inrabbit tibialis anterior muscle 40 min prior to plasmid EGT. Muscleswere collected 4 days p.i. and treated as described in materials andmethods section. The left muscle represents tibialis anterior fromrabbits pretreated with HYAse, the right muscle represents rabbitselectroinjected with plasmid DNA alone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of enhancing electrostimulationof a pharmaceutical agent, including but not limited to nucleicacid-based formulations, into vertebrate tissue which comprisesadministering a biologically effective amount of hyaluronidase (HYAse)in combination with an electro-gene transfer treatment.

In other words, the present invention improves upon previous techniqueswhich enhance delivery of the pharmaceutical agent by applyingelectrostimulation to points proximal to the site of injection (thus,electro-gene transfer, or “EGT”). In the present invention, applicationof hyaluronidase to (a) the area proximal to the site of administrationof the pharmaceutical agent and region of electrostimulation, or (b)simultaneous delivery of hyaluronidase and pharmaceutical agent(preferably in a single formulation or composition) in conjunction withapplication of electrostimulation, results in an increased transfer ofthe pharmaceutical agent (exemplified herein with DNA plasmidconstructions) as compared to application of the respectiveelectrostimulation protocol alone.

As noted herein, the present invention relates to a method of deliveringa pharmaceutical agent into a tissue of a vertebrate host whichcomprises the steps of administering a biologically effective amount ofhyaluronidase to the tissue of the vertebrate host; administering thepharmaceutical agent proximal to the delivery points of HYAseadministration and, applying an electrical stimulus proximal to the siteof administration of the hyaluronidase and the pharmaceutical agent. Tothis end, the methodology of the present invention relates to deliveryof a pharmaceutical agent, such as a population of nucleic acidmolecules (exemplified herein as DNA plasmid molecules), into the tissueof a vertebrate host, which comprises a) administering a biologicallyeffective amount of hyaluronidase to the tissue of the vertebrate host;b) administering the pharmaceutical agent proximal to the delivery siteof hyaluronidase administration in step a); and, c) applying anelectrical stimulus proximal to the delivery points of step a) and stepb). This methodology results in a substantial increase in delivery, andhence in vivo efficacy, of electrical-based delivery technology. It isshown herein that administration of HYAse may occur hours or minutesprior to electrical stimulation. Therefore, the methodology of thepresent invention logically covers a time spectrum regarding HYAseadministration from more than four hours up to minutes, or even inconjunction with electrostimulation of the target tissue. To this end,one aspect of the invention further relates to methodology disclosedherein whereby HYAse and the pharmaceutical agent (such as a DNA plasmidconstruct) are formulated together in order to allow for a present asingle injection at the target site.

To this end, given the exemplification that administration of HYAse mayoccur hours or minutes prior to electrical stimulation, a preferredaspect of this invention is a pharmaceutical formulation or compositionwhich comprises both an effective amount of hyaluronidase and therespective pharmaceutical agent. A preferred pharmaceutical agent is aneffective concentration of nucleic acid molecules, and most preferably abiologically effective concentration of DNA plasmid molecules. Such aformulation will be especially useful for dual administration of HYAseand the pharmaceutical agent in a scenario whereby a only a singleinjection is required in conjunction with EGT for transfer andexpression of nucleic acid-based vehicles.

The present invention therefore relates to formulations and methods ofenhancing EGT of nucleic acids into mammalian tissue which comprisesadministering a biologically effective amount of hyaluronidase (HYAse)in combination with administration of a pharmaceutical agent an EGTtreatment. As noted above, a preferred formulation may be a formulationwhich comprises both HYAse and a pharmaceutical agent, such as aneffective amounts of DNA plasmid molecules expressing a transgene ofinterest.

The present invention further relates to methods of enhancing EGT ofnucleic acids into mammalian muscle tissue which comprises administeringa biologically effective amount of hyaluronidase (HYAse) in combinationwith an EGT treatment.

Therefore, a preferred vertebrate target host is a mammal, and anespecially preferred target host includes but is not limited to humansand non-human primates, and may also include any non-human mammal ofcommercial or domestic veterinary importance.

Additionally, while one or more tissue types from the vertebrate hostmay be targeted for the synergistic EGT/HYAse methodology of the presentinvention, a preferred tissue type is muscle tissue, which has beenshown to be a viable target tissue for various electrostimulationprotocols involving gene therapy and/or gene vaccination applications.

Hyaluronidase is utilized in clinical applications. Therefore, thecombination of HYAse administration and muscle EGT constitutes anefficient manner in which to enhance to delivery and expression of genetherapy and/or genetic vaccination constructions in order to achievegreater therapeutic and/or prophylactic levels of gene expression withinthe target host. A particularly preferred application within the fieldof DNA vaccine technology is the delivery of a DNA vaccine which encodesone or more HIV antigens, including but not limited to HIV Gag, HIV Poland/or HIV Nef. Formulations which comprise both HYAse and one of theDNA vaccines listed herein comprise one preferred aspect of the presentinvention. The delivery to and expression from muscle tissue may beenhanced by combining the application of an EGT treatment andadministration of a biologically effective amount of HYAse, providingfor improved enhanced cellular-mediated immune responses upon hostadministration. An effect of the improved delivery, expression and/orimmunogencity of such DNA vaccines may be a lower transmission rate topreviously uninfected individuals (i.e., prophylactic applications)and/or reduction in the levels of the viral loads within an infectedindividual (i.e., therapeutic applications), so as to prolong theasymptomatic phase of HIV-1 infection. Therefore, the essence of thepresent invention relates to methodology which provides for an increasein the level of gene expression and/or immune response subsequent todelivery of a respective gene therapy or gene vaccination constructionto the muscle tissue of a vertebrate host. A series of preferred hostsinclude a mammalian host including but not limited to humans andnon-human primates, and also include any non-human mammal of commercialor domestic veterinary importance. The methodology, and concomitantformulations which comprise HYAse and the respective pharmaceuticalagent, may be applicable to any gene therapy target that relies on theexpression of a secreted protein that can exert its biological effectsystemically. Examples include but are not limited to gene therapytargets such as EPO, Factor VIII, Factor IX, Growth hormone, variouscytokines, and interferon.

It will be understood that any known methodology relating to EGT may beutilized in combination with HYAse to promote increased efficiency ofthat particular gene transfer methodology within the target host.Briefly, it is known that applied electrical stimulation can affectbiological tissues. Applied electrical fields can affect a rate ofdiffusion through tissues by advection, or may vary the extent to whichfluids diffuse into certain parts of the tissues. For example,electrical stimulation can increase permeability of a membrane when itis desired to infuse tissue with a substance through the membrane, andthe rate of diffusion is at least partly a function of permeability.Certain electrical or electromagnetic stimulation effects have beenexplained with reference to iontophoresis, electrophoresis andelectroporation. These terms involve different forms electrical effects.They may be considered different ways to interpret the results that arecaused by a given electrical potential, current or electromagneticfield. Depending on amplitude, polarity, frequency, spatial geometry andother parameters, a given field may produce a combination of sucheffects.

Iontophoresis and electrophoresis generally concern applying a directcurrent electric field in order to drive migration of positive andnegative ions by electrostatic attraction and repulsion toward and awayfrom an anode and cathode. Electric fields also tend to increase themobility of the ions generally. Iontophoresis typically involves causingpolar ions in a solution to migrate through an intact membrane such asthe skin. Electrophoresis concerns the migration of ions in a fluid orgel under the influence of a polar electric field (i.e., a field with atleast a direct current component).

Electroporation often involves a relatively higher power electric field,often applied briefly or pulsed. A field applied at sufficient amplitudeand/or for a sufficient duration can induce microscopic pores to form ina membrane. The pores are commonly called “electropores” and the processof forming them is called electroporation. Depending on the power andduration of the energy applied to a membrane, the pores may be larger orsmaller and may persist for a longer or shorter time. Preferably thepores persist temporarily, such as only during application of the field,and close or heal quickly. In this disclosure the term “electo-genetransfer” (EGT), “electrical stimulation” and/or “electrostimulation”,used interchangeably herein, is not limited to any one or any particularcombination of iontophoresis, electroporation, electrophoresis or anyother electrical effects. The terms as used herein are intended toencompass any such effects. A given electrical stimulation could haveresults that fall into more than one class, or possibly could bestronger in one or another due to the amplitude, polarity, spatialgeometry and/or timing involved. For example, a given direct current orlow frequency field could conceivably have sufficient amplitude toinduce pore formation (electroporation) while also causingelectrostatically driven ion migration through a membrane(iontophoresis) and accumulated migration with time (electrophoresis).Typically, however, electroporation involves higher electric fieldamplitudes than the other effects, and typically application at suchamplitudes is brief or intermittent or is pulsed at a duty cycle that issufficiently low to prevent unacceptable tissue damage.

The application of an electromagnetic field to tissue is complicated bythe fact that tissue is not homogeneous, isotropic or otherwise regularfrom an electromagnetic perspective. An applied field and an inducedcurrent can become concentrated by variations in the material propertiesof the tissue, including but not limited to the magnetic permeabilityand resistivity of tissues on a microscopic scale, and on a moremacroscopic scale, by the anatomical structure and organization oftissues.

Electrically induced pores have been observed and studied to a degree,in vitro, where cells in a solution are substantially independent of oneanother and are exposed to view. It is difficult or impossible toobserve the effects at a particular site in vivo. For example, obtainingaccess to tissue in vivo, such as sectioning the tissue to expose a siteto view, tends to disturb the tissue in ways that alter the localamplitude, orientation or other aspects of the applied electricalenergy. Thus it is difficult to make a meaningful in vivo observation ofelectrical stimulation parameters and effects.

Genetic and immunological therapies are candidates for electricalstimulation of tissues. Inasmuch as electrical stimulation tends toinvolve movement of ions and the opening of pores in tissues, it isplausible to apply a medicinal or other composition to a tissue site andto use electrical stimulation to move ions or molecules of thecomposition into positions, perhaps through pores in tissue membranes,where a desired effect is achieved or enhanced. Diffusion from thermaleffects (Brownian motion) could drive diffusion through electroporatedtissue membranes into an internal volume. Electrostatic or otherelectromagnetic effects could drive diffusion of ions through biologicalstructures, or at least increase the motion of affected molecules (e.g.,assuming an alternating polarity field), and thus affect particularreactions in order to achieve or to induce a therapeutic effect.

The limited gene transfer efficacy characteristic of plasmid DNAinjection has been ascribed, at least in part, to the presence ofabundant connective tissue, particularly in large animals, that mayprevent appropriate contact between the injected DNA and the musclefiber. This hypothesis is consistent with the idea that theextracellular matrix may play an important role in protecting musclefibers against penetration by exogenous molecules, bacteria, or virus.Although electrostimulation of muscle tissue may enhance DNA transfer byincreasing membrane permeability of muscle fibers, it is probable thatthe structural characteristic of the extracellular matrix may influencethe gene transfer efficiency of the injected DNA across the treatedmuscle. Thus, enzymatic permeabilization of the extracellular matrixcould create pores large enough to allow the productive interactionbetween the injected DNA and the muscle fiber. It is disclosed hereinthe effect on muscle EGT of extracellular matrix disruption by anenzymatic treatment (i.d., HYAse treatment) on muscle gene transfer andexpression. HYAse hydrolyzes hyaluronic acid which is a ubiquitaryconstituent of the extracellular matrix. Treatment of mouse and rabbitskeletal muscle with HYAse prior to DNA injection and electricalstimulation results in enhanced and long lasting gene expression withoutany significant tissue alteration. To this end, the present inventionrelates to the improvement of electrostimulation methodology byadministering a biologically effective amount of hyaluronidase (HYAse).The administration of HYAse will increase the transfer of the genetherapy and/or gene vaccination construction as compared to therespective electrostimulation parameters without administration ofHYAse. In other words, HYAse provides for a synergistic increase in genetransfer when utilized in conjunction with electrostimulationmethodology. As noted above, any such electrostimulation-basedmethodology contemplated by the skilled artisan to improve gene transfermay be utilized in conjunction with administration of HYAse to thetarget host. Variations in EGT parameters may be utilized in practicingthe present invention, including but not necessarily limited to varyingvoltage, the duration of pulse, the rotation of the electric field, thenumber of pulses, their frequencies, the interval between pulses, aswell as the timing of administration of the pharmaceutical agent andelectrostimulation. Examples of such techniques include but are notlimited to the following: U.S. Pat. No. 6,110,161 (see also Mathiesen,1999, Gene Therapy 6: 508-514) which discloses in vivo electricalstimulation of skeletal muscle within a calculated electric fieldstrength ranging from about 25 V/cm to about 250 V/cm; PCT Internationalpublications WO 99/01158, WO 99/01157 and WO 99/01175, which disclosedthe use of low voltage for a long duration to promote in vivo electricalstimulation of naked DNA, with an electric field strength or voltagegradient of about 1 V/cm to about 600 V/cm is disclosed; U.S. Pat. No.5,810,762, U.S. Pat. No. 5,704,908, U.S. Pat. No. 5,702,359, U.S. Pat.No. 5,676,646, U.S. Pat. No. 5,545,130, U.S. Pat. No. 5,507,724, U.S.Pat. No. 5,501,662, U.S. Pat. No. 5,439,440 and U.S. Pat. No. 5,273,525which disclose electroporation/electrostimulation methodology andrelated apparatus wherein it is suggested that a useful electrical fieldstrength range within the respective tissue is from about 200 V/cm toabout 20 KV/cm, while U.S. Pat. Nos. 5,968,006 and 5,869,326 furthersuggest that electric field strengths as low as 100 V/cm are useful forcertain in vivo electrostimulation procedures. Additional studies (withvarying parameters, as discussed in the Background of the Invention) canbe found, for example, in Titomirov et al.(1991, Biochem Biophys Acta1088; 131-134), Heller et al. (1996, FEBS Letters 389: 225-228), Nishiet al. (1996, Cancer Res. 56: 1050-1055), Zhang et al. (1996, Biochem.Biophys. Res. Comm. 220: 633-636), Muramatsu et al. (1997, Biochem.Biophys. Res. Comm. 223: 45-49), Rols et al. (1998, Nature Biotechnology16(2): 168-171), Aihara and Miyazaki (1998, Nature Biotechnology 16:867-870), Vicat et al. (2000, Human Gene Therapy 11: 909-916), Widera etal (2000, J. Immunology 164: 4635-4640), Suzuzki et al. (1998, FEBSLett. 425: 436-440), Goto et al. (2000, Proc. Natl. Acad. Sci. USA. 97:354-359), Oshima et al. (1998, Gene Therapy 5; 1347-1354), Rizzuto etal. (1999, Proc. Natl. Acad. Sci. USA 96: 6417-6422), Mir et al., (1998,C. R. Acad. Sci. Paris (Life Science) 321: 893-899), Mir et al, (1999;Proc. Nat. Acad. Sci. USA. 96: 4262-4267); Maruyama et al. (2000,Human-Gene Therapy 11: 429-437) and Draghia-Akli et al. (1999, NatureBiotechnology 17: 1179-1183). The following patent and non-patentpublications are hereby incorporated by reference so far as they pertainto known methodology for promoting gene transfer and expression thoroughelectrostimulation of respective target tissue.

It is shown herein that preinjecting hyaluronidase (HYAse) significantlyincreases the gene transfer efficiency of muscle EGT. Two constructsencoding mouse erythropoietin (pCMV/mEPO) and secreted alkalinephosphatase (pCMV/SeAP) were electro injected intramuscularly in Balb/Cmice and rabbits with and without HYAse pretreatment. Preinjection 1 or4 hr prior to EGT increased EPO gene expression by about 5 fold in miceand maintained higher gene expression than plasmid EGT alone. A similarincrement in gene expression was observed upon pretreatment with HYAseand pCMV/mEPO electroinjection in rabbit tibialis muscle. The incrementof gene expression in rabbits reached 17 fold upon injection of plasmidpCMV/SeAP. Injection of a plasmid encoding β-galactosidase(pCMV/βgal/NLS) and subsequent X-gal staining indicated that HYAseincreased the tissue area involved in gene expression. No irreversibletissue damage was observed upon histology analysis of treated mousequadriceps.

Therefore, the present invention relates to, as exemplified herein, theenhancement of transduction efficiency of EGT by injection of HYAse. Itis shown herein that a pretreatment with HYAse has long lasting effectsand increases gene expression by three to ten fold. This observation hasimportant implications for the development of a gene transfer protocolsuitable for therapeutic applications. Hyaluronidase catalyzes thehydrolysis of the β(14) linkage of hyaluronic acid, leading to itsdepolymerization and causing a temporary decrease in viscosity in theextracellular ground substance of the connective tissue. It is shownherein (FIG. 1A-B) that HYAse increases transduction efficiency in adose-dependent manner and is consistent with the mode of action of theenzyme (FIG. 2). Histology experiments with pCMV/NLS/β-gal indicate thatHYAse significantly enhances the tissue area involved in geneexpression. (FIG. 6). This may be due to an increase of DNA distributionthroughout the tissue, thus leading to an augmented bioavailability ofplasmid DNA. HYAse treatment may also contribute to releasing plasmidDNA from interactions with components of cellular matrix that mayinterfere with DNA entry upon electrical stimulation. HYAseadministration without muscle ES does not appear to increase genetransfer efficiency, even after injection of 100 μg of pCMV/mEPO. Thedata disclosed herein shows that HYAse does not directly influencecellular uptake of plasmid DNA but is dependent on muscleelectrostimulation to exert its effect on gene expression. Theseobservations distinguish the use of HYAse from sodium phosphate,recently reported as enhancing gene expression in muscle by inhibitingDNA degradation (Hartikka et al., 2000, Gene Therapy 7: 1171-1182.), aswell as non-ionic carriers such as polyvinyl pyrrolidone and SP1017(Lemieux et al., 2000, Gene Therapy 7: 986-991; Mumper et al., 1996,Pharm. Res. 13: 114-121; Alakhov et al., 1995; Bioconj. Chem. 7:209-216; Batrakova et al., 1996; Br J Cancer 74: 1545-1552). Theenhanced gene expression associated to HYAse treatment does notinfluence the overall stability of injected DNA as shown by theprogressive decline in EPO expression that is observed in all injectedanimals (FIG. 3A-C). Although it is likely that an enhanced DNA transferacross the treated muscle will guarantee a prolonged gene expression,this observation suggests that factors such as DNA stability andpromoter attenuation are to be considered important determinants of theefficacy of in vivo gene transfer. Hyaluronidase is currently utilizedfor clinical applications for such uses as the facilitation ofhypodermoclysis, the readsorption of edemas and in the formulation oflocal anesthetics. Thus, it is not surprising that histology analysisdid not reveal significant or permanent alterations of the injectedtissues (FIG. 4 A-C), and that animals injected with HYAse did not showany sign of discomfort. This is in contrast with risks of extensivemuscle damage associated with the use of potent muscle regeneratingagents such as cardiotoxin and bupivacain. A 17-fold increase inexpression was observed in rabbits upon injection of plasmid pCMV/SeAP,whereas EPO expression was augmented 3 fold (FIG. 5A-B). The reasons forthe differences in enhancement of gene expression between EPO and SeAPmay reside in the sensitivity of the detection assays as well as on thestability of the expressed proteins. Alternatively, this difference mayreflect a varying efficiency of secretion of EPO and SeAP from skeletalmuscle (e.g., see Kreiss et al., 1999, J. of Gene Med. 1: 245-250).Nonetheless, the significant increase in gene expression observed uponHYAse injection in rabbits indicates that the use of this enzyme couldguarantee increased gene transfer efficiency in large animals. Thisconclusion is particularly relevant for the more broad application ofmuscle EGT for human therapy, which will probably require a small numberof injections and a minimal amount of injected DNA along with asustained expression at therapeutic levels.

The nucleic acid molecules for use in the EGT/HYAse methodology of thepresent invention may be formulated in any pharmaceutically effectiveformulation for host administration; As noted throughout thisdisclosure, a preferred formulation is a formulation which comprisesboth HYAse and the respective pharmaceutical agent in biologicallyeffective concentrations. Any such formulation may be, for example, in asaline solution such as phosphate buffered saline (PBS). It will beuseful to utilize pharmaceutically acceptable formulations which alsoprovide long-term stability of the nucleic acid molecules, such as a DNAplasmid construction. During storage as a pharmaceutical entity, DNAplasmid molecules undergo a physiochemical change in which thesupercoiled plasmid converts to the open circular and linear form. Avariety of storage conditions (low pH, high temperature, low ionicstrength) can accelerate this process. Therefore, the removal and/orchelation of trace metal ions (with succinic or malic acid, or withchelators containing multiple phosphate ligands) from the DNA plasmidsolution, from the formulation buffers or from the vials and closures,stabilizes the DNA plasmid from this degradation pathway during storage.In addition, inclusion of non-reducing free radical scavengers, such asethanol or glycerol, are useful to prevent damage of the DNA plasmidfrom free radical production that may still occur, even in apparentlydemetalated solutions. Furthermore, the buffer type, pH, saltconcentration, light exposure, as well as the type of sterilizationprocess used to prepare the vials, may be controlled in the formulationto optimize the stability of the DNA molecule. Therefore, formulationsthat will provide the highest stability of the nucleic acid moleculesuch as a DNA plasmid vector will be one that includes a demetalatedsolution containing a buffer (phosphate or bicarbonate) with a pH in therange of 7-8, a salt (NaCl, KCl or LiCl) in the range of 100-200 mM, ametal ion chelator (e.g., EDTA, diethylenetriaminepenta-acetic acid(DTPA), malate, inositol hexaphosphate, tripolyphosphate orpolyphosphoric acid), a non-reducing free radical scavenger (e.g.ethanol, glycerol, methionine or dimethyl sulfoxide) and the highestappropriate DNA concentration in a sterile glass vial, packaged toprotect the highly purified, nuclease free DNA from light. Aparticularly preferred formulation which will enhance long termstability of the DNA vector vaccines of the present invention wouldcomprise a Tris-HCl buffer at a pH from about 8.0 to about 9.0; ethanolor glycerol at about 3% w/v; EDTA or DTPA in a concentration range up toabout 5 mM; and NaCl at a concentration from about 50 mM to about 500mM. The use of such stabilized DNA vector vaccines and variousalternatives to this preferred formulation range is described in detailin PCT International Application No. PCT/US97/06655 and PCTInternational Publication No. WO 97/40839, both of which are herebyincorporated by reference.

The nucleic acid molecules described herein may also be formulated withan adjuvant or adjuvants which may increase immunogenicity of the genetherapy or gene vaccination vehicle. A number of these adjuvants areknown in the art and are available for use in a DNA vaccine, includingbut not limited to particle bombardment using DNA-coated gold beads,co-administration of DNA vaccines with plasmid DNA expressing cytokines,chemokines, or costimulatory molecules, formulation of DNA with cationiclipids or with experimental adjuvants such as saponin, monophosphoryllipid A or other compounds which increase the efficacy of a particulargene therapy or gene vaccination construction. Another adjuvant for usein conjunction with the methodology disclosed herein are one or moreforms of an aluminum phosphate-based adjuvant wherein the aluminumphosphate-based adjuvant possesses a molar PO₄/Al ratio of approximately0.9. An additional mineral-based adjuvant may be generated from one ormore forms of a calcium phosphate. These mineral-based adjuvants areparticularly useful in increasing cellular and humoral responses to DNAvaccination. These mineral-based compounds for use as DNA vaccinesadjuvants are disclosed in PCT International Application No.PCT/US98/02414, PCT International Publication No. WO 98/35562, which ishereby incorporated by reference. Another preferred adjuvant is anon-ionic block copolymer which shows adjuvant activity with DNAvaccines. The basic structure comprises blocks of polyoxyethylene (POE)and polyoxypropylene (POP) such as a POE-POP-POE block copolymer. Newmanet al. (1998, Critical Reviews in Therapeutic Drug Carrier Systems15(2): 89-142) review a class of non-ionic block copolymers which showadjuvant activity. The basic structure comprises blocks ofpolyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-POP-POEblock copolymer. Newman et al. id., disclose that certain POE-POP-POEblock copolymers may be useful as adjuvants to an influenzaprotein-based vaccine, namely higher molecular weight POE-POP-POE blockcopolymers containing a central POP block having a molecular weight ofover about 9000 daltons to about 20,000 daltons and flanking POE blockswhich comprise up to about 20% of the total molecular weight of thecopolymer (see also U.S. Reissue Patent No. 36,665, U.S. Pat. No.5,567,859, U.S. Pat. No. 5,691,387, U.S. Pat. No. 5,696,298 and U.S.Pat. No. 5,990,241, all issued to Emanuele, et al., regarding thesePOE-POP-POE block copolymers). WO 96/04932 further discloses highermolecular weight POE/POP block copolymers which have surfactantcharacteristics and show biological efficacy as vaccine adjuvants. Theabove cited references within this paragraph are hereby incorporated byreference in their entirety. It is therefore within the purview of theskilled artisan to utilize available adjuvants which may increase theimmune response of the polynucleotide vaccines of the present inventionin comparison to administration of a non-adjuvanted polynucleotidevaccine.

The EGT/HYAse methodology of the present invention may call for theadministration of either/or of the nucleic acid construction and HYAseby any means known in the art, such as enteral and parenteral routes.The preferred route of administration is intramuscular. Additionalroutes included but are not limited to subcutaneous administration,intraperitoneal injection, intravenous injection, inhalation orintranasal delivery, oral delivery, sublingual administration,transdermal administration, transcutaneous administration, percutaneousadministration or any form of particle bombardment, such as a biolisticdevice such as a “gene gun” or by any available needle-free injectiondevice. The preferred method of delivery of the nucleic acidconstruction and HYAse intramuscular injection via needle in conjunctionwith a respective EGT protocol. Additional methods of delivery includebut are not necessarily limited to subcutaneous administration andneedle-free injection. A particular mode of administration is the use ofa sort of ointment, as noted above.

The amount of expressible DNA to be introduced to a host recipient willdepend on the strength of the transcriptional and translationalpromoters used in the DNA construct, and on the level of expressedprotein required to treat the disease or disorder, or on theimmunogenicity of the expressed gene product. In general, an effectivedose of about 1 μg to greater than about 20 mg, and preferably in dosesfrom about 1 mg to about 5 mg is administered directly into muscletissue. As noted above, subcutaneous injection, intradermalintroduction, impression through the skin, and other modes ofadministration such as intraperitoneal, intravenous, inhalation and oraldelivery are also contemplated. It is also contemplated that boosterapplications may be utilized, which will again be construct and diseasespecific, so as to optimize the effectiveness of the gene therapy orgene vaccination application.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1

Plasmid preparation—Constructs pCMV/mEPO, pCMV/β-gal/NLS, and pCMV/SeAPwere constructed as follows: The complete mouse EPO (mEPO) codingregion, including 40 bp of the 5′ untranslated region (Shoemaker andMitsock, 1986, Mol. Cell Biol. 6: 849-858) was assembled from syntheticoligonucleotides as described (Stemmer et al., 1995, Gene 164: 49-53),with minor modifications. Briefly, 15 oligos, 60 nt in length, were usedafter gel purification: 8 oligos covered part of the sequence of theplus strand, whereas 7 oligos covered part of the sequence of the minusstrand, and the oligos were configured in such a way that, uponassembly, they overlapped with regions of complementarity of 20 nt. TwoSacI sites and two PstI sites present in the mEPO coding sequence wereeliminated without altering the encoded protein sequence and, at thesame time, optimizing the codon usage. Gene assembly was performed asdescribed (Stemmer et al., 1995, id.), and the entire coding region wasverified by dideoxy sequencing. Plasmid pCMV/mEPO was constructed byinserting the mEPO coding sequence as a EcoRI-BamHI 0.6 Kb fragment intopViJnsB (Montgomery et al. 1993, DNA Cell. Biol. 12: 777-783) containingthe CMV immediate/early region promoter and enhancer with intron Afollowed by the BGH polyadenylation signal. To construct pViJ/β-gal/nlsa 3.5 Kb BamHI β-gal/nls encoding fragment was excised from pGM48β-gal(Wiznerowicz et al., 1997) and cloned in the BglII restriction site ofpViJnsB (Montgomery et al., id). Plasmid pViJ/SEAP was constructed byinserting the coding sequence of secreted alkaline phosphatase in theBglII restriction site of pViJnsB (Montgomery et al., id.).

Plasmid pCMV/mEPOopt carries the mouse EPO cDNA codon-optimized tomammals. Briefly, the EPO cDNA coding sequence was modified such thatthe native codons were substituted with codons frequently found inhighly expressed human genes. Th optimized sequence of mouse EPO is asfollows: ATGGGCGTGC CCGAGCGCCC CACCCTGCTG CTGCTGCTGA GCCTGCTCCTGATCCCCCTG (SEQ ID NO:1) GGCCTGCCCG TGCTGTGCGC CCCCCCCCGC CTGATCTGCGACAGCCGCGT GCTGGAGCGC TACATCCTGG AGGCCAAGGA GGCCGAGAAC GTGACCATGGGCTGCGCTGA GGGCCCCCGC CTGAGCGAGA ACATCACCGT GCCCGACACC AAGGTGAACTTCTACGCCTG GAAGCGCATG GAGGTGGAGG AGCAGGCCAT CGAGGTGTGG CAGGGCCTGTCCCTGCTGTC TGAGGCCATC CTGCAGGCCC AGGCCCTGCT GGCCAACTCC TCCCAGCCCCCCGAGACCCT GCAGCTGCAC ATCGACAAGG CCATCAGCGG CCTGCGCTCC CTGACCTCCCTGCTGCGCGT GCTGGGCGCC CAGAAGGAGC TGATGAGCCC CCCCGACACC ACCCCCCCCGCCCCCCTGCG CACCCTGACC GTGGACACCT TCTGCAAGCT GTTCCGCGTG TACGCCAACTTCCTGCGCGG CAAGCTGAAG CTGTACACCG GCGAGGTGTG CCGCCGCGGC GACCGCTGA.Plasmid DNA was prepared by standard double CsCl gradient purificationand resuspended in sterile saline solution.

Animals and treatment—Six week old female Balb/C mice and 10 week oldfemale rabbits were purchased from Charles River Breeding Laboratory andused in all experiments. Animals were maintained in standard conditionsunder 12-hr light-dark cycle, provided irradiated food and chlorinatedwater ad libitum. All animal procedures were conducted in conformitywith national and international laws and policies.

Electro-gene transfer—Mouse quadriceps and rabbit tibialis anteriormuscle were surgically exposed and injected with a predetermined amountof plasmid DNA. The volume of injection was kept constant at 50 μl inmice and 200 μl in rabbits. Where indicated, hyaluronidase wasresuspended in 50 μl (mice) or 500 μl (rabbits) of sterile salinesolution at the desired concentration and injected prior to EGT at theindicated time. HYAse may be purified as disclosed by Borders, et al.,1965, J. Biol. Chem. 243: 3750-3762. For these studies, HYAse wasobtained from Sigma (St. Louis. MO); Type: VI-S from Bovine Testes;Enzyme Commission Number: 3.2.1.35; Synonyms: Hyaluronoglucosamidase,Hyaluronate 4-glycanohydrolase; Source: Bovine testes). Steel electrodesin the form of parallel 0.2 mrn wires about 3 cm long and 5 mm apartwere inserted intramuscularly around the injection site. The electricfield was applied in a pulsed form as described (Rizzuto et al., 1999,Proc. Natl. Acad. Sci. 96:6417-6422) with minor modifications. Briefly,mouse quadriceps muscles were surgically exposed and injected with apredetermined amount of plasmid DNA. Steel electrodes in the form ofparallel 0.2 mm wires about 3 cm long and 5 mm apart were brought intocontact with the muscle in parallel orientation with respect to themuscle fibres. The electric field was applied in a pulsed form through aPulsar 6 bp-a/s bipolar stimulator (FHC, ME, USA) and each cycle ofstimulation comprised a one second pulse train of square bipolar pulsesdelivered every other second. Each train consisted in 10³ pulses of 200μsec length and 45 Volts amplitude. Pulses were monitored using adigital oscilloscope. A custom amplifier was constructed using an APEXPA-85 power operational amplifier in the output stage (APEXTechnologies, Tucson, Ariz.). Signals were generated by an integratedcustom signal generator and were monitored using a two-channel 8-bitoscilloscope card (K7103 Velleman, Gavere, Belgium). The entire set upwas controlled by a custom software package written in Java programminglanguage running on PC-compatible laptop (Extensa 501T, Acer America,San Jose, Calif.). Voltage and current were measured periodically duringthe experiment with a digital oscilloscope. Voltage was monitored acrossthe lower resistor of a voltage divider (100,000 ohms resistor over a10,000 ohm resistor) in parallel with the electrodes, whereas currentwas monitored by measuring the potential drop across a precision 1 ohmresistor in series with the electrodes.

Histological analysis—Mouse quadriceps and rabbit tibialis were removedat the indicated time after treatment and fixed 3 h in ice in 0.50%glutaraldehyde and 2% paraformaldehyde in sodium phosphate buffer (pH7.4) containing 0.02% Nonidet P-40. After three washes in ice cold PBSmuscles were incubated in a reaction mixture containing 2 mM 5-bromo-4chloro-3-indolyl-β—D-galactosidase (X-gal, GIBCO BRL), 2 mM MgCl₂, 4 mMpotassium ferricyanide, 4 mM potassium ferrocyanide, 0.02% Nonidet P40in sodium phosphate buffer at 30° C. overnight. After incubation,quadriceps were washed three times in PBS and embedded in 20% sucrose insodium phosphate buffer (pH 7.4) for 5 hrs at 4° C. Cryostatic musclesections were finally examined for β-galactosidase expression by lightmicroscopy. For the assessment of tissue damage and morphology, tissueswere embedded in paraffin, stained with Haematoxylin-Eosin (H/E) andexamined under light microscope as described (Ausubel et al., 1992).

Hematocrit, EPO, and SeAP measurement—Blood samples were collected atthe indicated times. Hematocrits were determined by centrifugation ofwhole blood in heparinized capillary tubes as previously described(Rizzuto et al., 1999, Proc. Natl. Acad. Sci. 96:6417-6422). Serum SeAPlevels were monitored by Phospha-Light (Chemiluminescent Reporter Assayfor secreted alkaline phosphatase), Tropix. Results were analyzed byusing ANOVA analysis (STAT-VIEW, Abacus Concept Berkeley Calif.). A Pvalue <0.05 was considered significant.

Results—Effect of Hyaluronidase on EPO gene expression in mouse muscle.To assess the effect of HYAse on EPO gene expression, the quadricepsmuscle of groups of 4 Balb/c mice were injected with different amountsof HYAse ranging from 0.5 to 90 U. Four hours later, 3 μg of plasmidpCMV/mEPO were injected in the same muscle and the treated tissue wassubjected to ES as previously described above. EPO levels and Hct valuesof treated mice were determined one week after injection and compared tothose of a control group that did not receive HYAse and was injectedwith the same amount of pCMV/mEPO. As shown in FIG. 1A, increase of EPOvalues in DNA injected and muscle ES mice was significant over that ofsaline treated animals (121.8 mU/ml) (1 mU=10 pg). Pre injection of 90 Uof HYAse resulted in a five-fold increase in EPO expression (550 mU/ml).Similarly, pre injection of 36 U of HYAse also resulted in aconsiderable increase in EPO levels, albeit slightly lower than thatobserved with 90 U (455 mU/ml). In contrast, injection of 18, 5.4, 1.8,or 0.5 U of HYAse did not result in a substantial increase in EPO levelsas compared to injection of plasmid DNA alone. No increase in EPO levelswas observed upon injection of HYAse and up to 100 μg of plasmidpCMV/mEPO in mice that were not subjected to ES.

The increase in serum EPO levels resulted in a notable increment in theHct of treated animals as compared to saline treated controls (FIG. 1B).However, at this time point, the measured Hct values did notsignificantly differ among the different treated groups. The lack of aquantitative difference between the Hct values of the various micegroups at this time point simply reflects the notion that erythropoiesisis regulated by a series of factors in addition to EPO that can limitthe progression of Hct increase. Nonetheless, these results indicatethat pre-injection of HYAse leads to an increase in EPO gene expressionupon DNA injection and muscle ES.

To determine the pre-injection time for HYAse administration requiredfor maximal EPO gene expression, groups of Balb/c mice were injectedwith HYAse 4 h, 1 h, and 10 minutes prior to EGT. The amount of HYAseinjected into the mouse quadriceps was fixed at 36 U, since itcorresponds to 720 U/ml and is within the range of HYAse used forclinical applications (150 to 1500 U; Berger, 1984, J. Am. Geriatr.Soc.32: 199-203). As shown in FIG. 2, measurement of EPO levels one-weekpost injection (p.i.) indicated that HYAse injection 4 or 1 hr and 10min prior to DNA injection and muscle ES resulted in 5-fold increase inserum EPO level as compared to that of mice injected with DNA alone.Thus, these data demonstrate that administration of HYAse 10 min priorto electroinjection of plasmid is sufficient to lead to a significantincrease in EPO gene expression in mice.

Long term effect of HYAse injection on EPO gene expression—Verificationas to whether the increase of EPO gene expression upon HYAseadministration could be observed over time and independently of DNAdosage was undertaken. To this end, groups of animals were injected withdifferent amounts of plasmid pCMV/mEPO and serum EPO levels of mice thathad been pre injected with HYAse were measured over time and compared tothose of mice treated with DNA alone (FIG. 3A-C). Serum EPO levelsmeasured at 7, 56, and 120 days p.i. correlated with the amount ofinjected DNA. The EPO values observed in mice that had been pretreatedwith HYAse were consistently higher than those of animals treated withDNA alone and ranged from 64 mU/ml with 0.5 μg of DNA to 1324 mU/ml atday 7 in mice injected with 50 μg of DNA. Groups pretreated with HYAseand injected with 3, 10, 50 μg of plasmid DNA showed EPO levels thatwere significantly different from those detected in mice electroinjectedwith DNA alone. In all treated groups, circulating EPO reached a peaklevel at 7 days p.i. (FIG. 3A), decreased variably in the differentgroups to from ½ to ⅛ of the initial value after 56 days (FIG. 3B), andremained constant thereafter. Additionally, because of the high level ofEPO expression, mice injected with 50 μg of plasmid DNA after HYAseadministration displayed extremely high Hct values (>85%) and died by120 days p.i. (FIG. 3C). These results demonstrate that injection ofHYAse results in enhanced EPO expression independently of DNA dosage andthat such effect persists for a prolonged period of time.

Analysis of tissue damage—The extent of tissue alterations that could beassociated to the use of HYAse for EPO gene transfer was assessed.Histology analysis of HYAse injected quadriceps muscles was performed 1,3, 7, and 30 days p.i. No tissue alteration was detected 24 hrs afterinjection. The most striking and consistent pathological findings wereobserved only in samples 3 days after the treatment (FIG. 4A). In thesesamples lesions were detected in about the 20% of the total muscle mass.In paraffin embedded sections stained with H/E, areas of massivecolliquative necrosis of the muscle fibers were observed, in eachnecrotic area mononuclear cell infiltrates, mostly of macrophagicorigin, were detected. Each area was typically surrounded by a reactivefibrosis. Similar and consistent necrotic lesions were observed also insamples at 7 days after treatment but in this case they representedroughly 1% the total muscle mass (FIG. 4B). After 7 days no fibroticreaction was observed and mononuclear cells infiltrate were lessapparent. The necrotic lesions after 1 month were sporadic and less than1% of the muscle mass was involved. Additionally, mononuclear cellinfiltrates were no longer detected (FIG. 4C). Therefore, these findingssuggest that HYAse treatment results in limited and transient tissuedamage.

Effect of HYAse on gene transfer in large muscles—To assess the effectof HYAse administration on EPO gene expression of large muscles, aseries of DNA injection experiments were carried out on rabbit tibialisanterior muscle. Construct pCMV/mEPOopt was utilized for these studies.This plasmid carries a mouse EPO cDNA codon-optimized to mammals.Codon-optimized EPO constructs have been reported to express higher EPOlevels (Kim et al., 1997, Gene 199: 293-301). Additionally, to verifythat the effects of HYAse were not limited to EPO gene expression,rabbits were injected with a plasmid encoding secreted alkalinephosphatase (pCMV/SeAP) (Bettan et al, 1994, Anal. Biochem. 271:187-189). As shown in FIG. 5A-B, treatment with 180 U of HYAse 40 minprior to injection of 200 μg of pCMV/mEPOopt and ES resulted into a 3fold increase in EPO expression (FIG. 5A). The amount of HYAse injectedand time of injection were those that yielded the greater level of EPOexpression. A 17-fold increase in expression was detected in HYAsetreated rabbits upon injection of 200 μg of pCMV/SEAP and muscle ES(FIG. 5B). The same increment of SEAP expression was also noted oninjection of 0.5 and 1 mg of plasmid DNA. These results confirm theeffect of HYAse on gene expression observed in mice and demonstrate thatefficiency of gene expression is increased upon DNA injection and muscleES in large animals.

Effect of HYAse on β-galactosidase gene expression in muscle—To analyzethe effects of HYAse on tissue distribution of gene expression followingEGT of plasmid DNA, the β-galactosidase gene was utilized. Two hundredmicrograms of plasmid pCMV/β-gal/NLS encoding the E. coli lacZ fused toa nuclear localization signal were injected into the tibialis muscle 40mins after HYAse administration. The treated muscles were subjected toEGT. Additionally, the extent of β-gal expression was compared to thatof rabbits treated with EGT alone. The histology analysis demonstratedthat the area of positive X-gal staining was significantly larger inrabbits pretreated with HYAse than those in animals treated with DNAalone (FIG. 6). These results indicate that HYAse promotes distributionof plasmid DNA across the tissue.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method of delivering a pharmaceutical agent into a tissue of avertebrate host, which comprises: a) administering a biologicallyeffective amount of hyaluronidase to the tissue of the vertebrate host;b) administering the pharmaceutical agent proximal to the delivery siteof hyaluronidase administration of step a); and, c) applying anelectrical stimulus proximal to the delivery points of step a) and stepb), such that the amount of biologically effective pharmaceutical agentdelivered to the tissue of the vertebrate host is greater thanapplication of an electrical stimulus and pharmaceutical agent alone. 2.A method of claim 1 wherein the vertebrate host is a mammalian host. 3.A method of claim 2 wherein the mammalian host is a non-human primate.4. A method of claim 2 wherein the mammalian host is a human.
 5. Amethod of claim 4 wherein human muscle tissue of the human is targetedfor delivery of the pharmaceutical agent.
 6. A method of claim 5 whereinthe human muscle tissue is skeletal muscle tissue.
 7. A method of claims1, 2, 3, 4, 5, or 6 wherein the pharmaceutical agent is a nucleic acidmolecule.
 8. The method of claim 7 wherein the nucleic acid molecule isa DNA plasmid molecule.
 9. A method of delivering a pharmaceutical agentinto a tissue of a vertebrate host, which comprises: a) administering abiologically effective amount of hyaluronidase to the tissue of thevertebrate host up to about 4 hours prior to application of anelectrical stimulus; b) administering the pharmaceutical agent proximalto the delivery site of hyaluronidase administration of step a); and, c)applying the electrical stimulus proximal to the delivery points of stepa) and step b), such that the amount of biologically effectivepharmaceutical agent delivered to the tissue of the vertebrate host isgreater than application of an electrical stimulus and pharmaceuticalagent alone.
 10. A method of claim 9 wherein the vertebrate host is amammalian host.
 11. A method of claim 10 wherein the mammalian host is anon-human primate.
 12. A method of claim 10 wherein the mammalian hostis a human.
 13. A method of claim 12 wherein human muscle tissue of thehuman is targeted for delivery of the pharmaceutical agent.
 14. A methodof claim 13 wherein the human muscle tissue is skeletal muscle tissue.15. A method of claims 9, 10, 11, 12, 13, 14 or 15 wherein thepharmaceutical agent is a nucleic acid molecule.
 16. The method of claim15 wherein the nucleic acid molecule is a DNA plasmid molecule.
 17. Amethod of delivering a pharmaceutical agent into a tissue of avertebrate host, which comprises: a) administering a biologicallyeffective amount of hyaluronidase to the tissue of the vertebrate hostup to about 4 hours prior to application of an electrical stimulus; b).administering the pharmaceutical agent proximal to the delivery situreof hyaluronidase administration of step a); and, c) applying theelectrical stimulus proximal to the delivery points of step a) and stepb), such that the amount of biologically effective pharmaceutical agentdelivered to the tissue of the vertebrate host is greater thanapplication of an electrical stimulus and pharmaceutical agent alone,wherein the hyaluronidase of step a) and the pharmaceutical agent ofstep b) comprise a single formulation, said formulations beingadministered prior to or in conjunction with the application of theelectrical stimulus of step c).
 18. A method of claim 17 wherein thevertebrate host is a mammalian host.
 19. A method of claim 18 whereinthe mammalian host is a non-human primate.
 20. A method of claim 18wherein the mammalian host is a human.
 21. A method of claim 20 whereinhuman muscle tissue of the human is targeted for delivery of thepharmaceutical agent.
 22. A method of claim 21 wherein the human muscletissue is skeletal muscle tissue.
 23. A method of claims 17, 18, 19, 20,21, or 22 wherein the pharmaceutical agent is a nucleic acid molecule.24. The method of claim 23 wherein the nucleic acid molecule is a DNAplasmid molecule.
 25. A method of claims 1, 9 or 17 whereinhyaluronidase is administered by direct needle injection.
 26. A methodof claim 1 wherein the hyaluronidase of step a) is added from about 30minutes to 2 hours to application of the electrical stimulus of step c).27. A method of claim 1 wherein the hyaluronidase of step a) is addedfrom about 15 minutes to 45 minutes to application of the electricalstimulus of step c).
 28. A formulation which comprises hyaluronidase anda pharmaceutical agent.
 29. A formulation of claim 28 wherein thepharmaceutical agent is a nucleic acid molecule.
 30. A formulation ofclaim 29 wherein the nucleic acid molecule is a DNA plasmid molecule.